2′-Substituted RNA preparation

ABSTRACT

A process for the preparation of a compound of formula (1): 
                 
 
is provided, which comprises the reaction a compound of formula (2): 
                 
 
with a compound of formula Al(OR) 3  under substantially anhydrous conditions. X, and X 1  are each independently H or a protecting group, B is a base; R is an alkyl, alkoxyalkyl, alkenyl or alkynyl group, each of which may be optionally substituted, and L is a leaving group.

This application is the National Phase of International ApplicationPCT/GB00/00965 filed Mar. 15, 2000 which designated the U.S. and thatInternational Application was published under PCT article 21(2) inEnglish.

The present invention relates to a process for preparing2′-O-substituted nucleosides, and more particularly to a process for thepreparation of 2′-O-substituted uridine and cytidine.

The possibility that synthetic oligonucleotides might be effectiveinhibitors of gene expression and be used as chemotherapeutic agents hasstimulated much research work in recent years. In order to avoid theirdegradation by cellular nucleases, it is essential that sucholigonucleotides should be modified. Modifications can be made to theinternucleotide linkages, the base residues and the sugar residues. Alarge number of oligonucleotide analogues in which the internucleotidelinkages have been modified, especially as phosphorothioates withnon-bridging sulphur atoms, have been described. Several of thesephosphorothioate analogues are promising drug candidates that are nowundergoing clinical trials. However, phosphorothioates do have somedisadvantages. Thus, they do not display optimal RNA-binding propertiesand they also have a tendency to bind to proteins in a non-specificmanner. Possible base modifications are clearly limited as they must notlead to a significant decrease in hybridisation properties. Recently,considerable interest has been directed towards the modification of thesugar residues. One particular type of modification involves theintroduction of 2′-α-alkoxy groups (as in2′-O-alkyl-oligoribonucleotides). While, in general, small alkoxy groups(such as methoxy) promote better hybridisation properties withcomplementary ribonucleic acids (RNA), nuclease resistance tends toincrease with an increase in the size of the alkoxy group.2-Methoxyethoxy has emerged as an alkoxy group that confers both goodhybridisation properties and high nuclease resistance. It thereforeseems likely that 2′-O-(2-methoxyethyl)-ribonucleosides will beincorporated into a second generation of potential oligonucleotidechemotherapeutic agents. For this reason, the development of convenientmethods for the preparation of 2′-O-(2-methoxyethyl)-ribonucleosides hasbecome a matter of much importance.

The preparation of 2′-O-(2-methoxyethyl)-ribonucleosides, starting fromD-ribose, has previously been described. These preparations involved theuse of protecting groups and required a relatively large number ofsteps. For example, 2′-O-(2-methoxyethyl)-5-methyluridine was preparedby Martin, P. Helv. Chim. Acta 1995, 78, 486-504 from D-ribose in 10steps and in 33% overall yield. A later report by McGee and Zhai inAbstracts of American Chemical Society National Meeting, Division ofOrganic Chemistry, March 1996 paper 253 revealed a much more convenientprocedure for the preparation of 2′-O-alkyl derivatives of the mainpyrimidine ribonucleosides. Thus, when5′-O-(4,4′-dimethoxytrityl)2,2′-anhydro-1-β-D-arabinofuranosyluracil washeated with magnesium methoxide in N,N-dimethylformamide (DMF) at 100°C., 5′-O-(4,4′-dimethoxytrityl)-2′-O-methyluridine was obtained in 94%yield. Somewhat lower yields of the corresponding 5′O-ethyl-,5′-O-(n-propyl)- and 5′-O-allyl-uridine derivatives were obtained in thereactions between the same substrate and the appropriate magnesiumalkoxides. It was also reported that magnesium alkoxides could bereplaced by calcium alkoxides.

Ross et al reported in Nucleosides and Nucleotides, 1997, 16, 1641-3that when unprotected 2,2′-anhydro-1-β-D-arabinofuranosyluracil washeated with a twofold excess of trimethyl borate and a stoichiometricquantity of trimethyl orthoformate in methanol at 150° C., underpressure, for 42 h, 2′-O-methyluridine was obtained in 86% isolatedyield. 2′-O-Methyl-5-methyluridine was similarly prepared from2,2′-anhydro-5-methyl-(1-β-D-arabinofuranosyluracil) by the borate esterprocedure and, although no experimental details were provided, thepreparation of 2′-O-methylcytidine was also reported. The yield of2′-O-alkyl-uridine was stated to decrease with increasing alcohol size.

It remains desirable to identify additional or alternative routes forthe preparation of 2′-O-substituted nucleosides.

According to the present invention, there is provided a process for thepreparation of a compound of formula (1):

wherein:

-   -   X, and X¹ are each independently H or a protecting group;    -   B is a base; and    -   R is an alkyl, alkoxyalkyl, alkenyl, or alkynyl group, each of        which may be optionally substituted;        which comprises reacting a compound of formula (2):        wherein    -   L is a leaving group; and    -   B, X and X¹ are as defined above        with a compound of formula Al(OR)₃ wherein R is as defined        above, under substantially anhydrous conditions.

When R is alkenyl, the alkenyl group is often a C₁₋₄ alkenyl group,especially an allyl or crotyl group. When R represents alkyl, the alkylgroup is preferably a C₁₋₄ alkyl, and most preferably a methyl or ethylgroup. When R represents alkoxyalkyl, the alkoxyalkyl group is often aC₁₋₄ alkyoxyC₁₋₄ alkyl group, and preferably a methoxyethyl group. WhenR is alkynyl, the alkynyl group is often a C₁₋₄ alkynyl group,especially a propargyl group. The alkyl, alkenyl, alkynyl andalkoxyalkyl groups may themselves be substituted by one or moresubstituents, particularly halogen, especially F, Cl or Br, and aminosubstituents.

Examples of protecting groups which can be represented by X and X¹include acid labile protecting groups, particularly trityl andsubstituted trityl groups such as dimethoxytrityl and9-phenylxanthen-9-yl groups; acid-labile acetal protecting groups,particularly 1-(2-fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp); andbase labile-protecting groups such as acyl groups, commonly comprisingup to 16 carbon atoms, such as ethanoyl groups or fatty alkanoyl groups,including particularly linear or branched C₆₋₁₆ alkanoyl groups, such aslauroyl groups; benzoyl and substituted benzoyl groups, such as alkyl,commonly C₁₋₄ alkyl-, and halo, commonly chloro or fluoro, substitutedbenzoyl groups.

Other suitable protecting groups include those derived from gamma ketoacids, such as levulinoyl groups and substituted levulinoyl groups.Substituted levulinoyl groups include 5-halo-levulinoyl, such as5,5,5-trifluorolevulinoyl and benzoylpropionyl groups; and silyl andsiloxane ethers, such as alkyl, commonly C₁₋₄, alkyl, and aryl, commonlyphenyl, silyl ethers, particularly trialkylsilyl groups, oftentri(C₁₋₄-alkyl)silyl groups, such as tertiary butyl dimethyl silyl andtertiary butyl diphenyl silyl groups.

Bases which can be represented by B include nucleobases, particularlypurines, especially adenine (A) and guanine (G); and pyrimidines,especially thymine M, cytosine (C), and uracil (U); and substitutedderivatives thereof. Examples of substituents which may substitute thebases, in addition to protecting groups, include alkyl, especiallyC₁₋₄-alkyl, particularly methyl; halogen, particularly Cl or Br; amino;alkenyl, especially C₁₋₄-alkenyl and particularly allyl; alkoxyalkyl,especially C₁₋₄alkoxyC₁₋₄alkyl, particularly methoxyalkyl; and alkynyl,particularly propargyl, substitutents. The alkyl, alkenyl, alkynyl andalkoxyalkyl groups may themselves be substituted by one or moresubstituents, particularly halogen, especially F, Cl or Br, and aminosubstituents.

In addition to the presence of protecting groups X and X¹, basesemployed in present invention may also be protected where necessary bysuitable protecting groups. Protecting groups employed are those knownin the art for protecting such bases. For example, A and/or C can beprotected by benzoyl, including substituted benzoyl, for example alkyl-or alkoxy-, often C₁₋₄ alkyl- or C₁₋₄alkoxy-, benzoyl; pivaloyl; andamidine, particularly dialkylaminomethylene, preferably di(C₁₋₄-alkyl)aminomethylene such as dimethyl or dibutyl aminomethylene. G may beprotected by a phenyl group, including substituted phenyl, for example2,5-dichlorophenyl and also by an isobutyryl group. T and U generallyare not protected, but in certain embodiments they may advantageously beprotected, for example at O4 by a phenyl group, including substitutedphenyl, for example 2,4-dimethylphenyl or at N3 by a pivaloyloxymethyl,benzoyl, alkyl or alkoxy substituted benzoyl, such as C₁₋₄ alkyl- orC₁₋₄ alkoxybenzoyl.

In certain embodiments, X and X¹ comprise a single protecting groupwhich protects both the 3′ and 5′ positions. Examples of such groupsinclude disiloxanes, especially tetraalkyldisiloxanes, such astetraisopropyldisiloxane.

Leaving groups which can be represented by L include those leavinggroups which can be displaced by a nucleophile of formula RO⁻. Examplesof preferred leaving groups include groups of formula —OSO₂CH₃,—OSO₂CF₃, Cl, Br, I, O-Mesyl, O-Brosyl and O-Tosyl groups.

In certain preferred embodiments, the leaving group comprises the base,B, chemically bonded to the 2′-position, commonly via an oxygen orsulphur atom or a group of formula —NR^(X)—, wherein R^(X) is H or aC₁₋₆ alkyl or aryl, such as a phenyl, group. Most preferably, the baseis uracil bonded to the 2′-position via an oxygen atom.

Accordingly, a second aspect of the present invention provides a processfor the preparation of a compound of formula (3):

wherein:

-   X and X¹ are as defined above;-   R¹ and R² are each independently H, alkyl, alkenyl, alkynyl, or    halogen; and-   R is an alkyl, alkoxyalkyl, alkenyl, or alkynyl group, each of which    may be optionally substituted;    which comprises the reaction of a compound of formula (4)    wherein-   X, X¹, R¹ and R² are as defined above;-   with a compound of formula Al(OR)₃ wherein R is as defined above,    under substantially anhydrous conditions.

When either of R¹ and R² is alkenyl, the alkenyl group is often a C₁₋₄alkenyl group, especially an allyl or crotyl group. When either of R¹and R² represents alkyl, the alkyl group is preferably a C₁₋₄ alkyl, andmost preferably a methyl or ethyl group. When either of R¹ and R²represents alkoxyalkyl, the alkoxyalkyl group is often a C₁₋₄alkyoxyC₁₋₄ alkyl group, and preferably a methoxyethyl group. Wheneither of R¹ and R² is alkynyl, the alkynyl group is often a C₁₋₄alkynyl group, especially a propargyl group. The alkyl, alkenyl andalkynyl groups represented by R¹ or R² may be substituted by one or moresubstituents, particularly halogen, especially F, Cl or Br, and aminosubstituents. When either of R¹ and R² is halogen the halogen ispreferably Cl, Br or I. Most preferably, both of R¹ and R² represent H,or R¹ represents C₁₋₄ alkyl and R² represents H.

The process according to the present invention takes place in thepresence of a suitable substantially anhydrous solvent. Examples ofsuitable solvents include halocarbons such as chloroform,1,2-dichloroethane and chlorobenzene; esters, particularly alkyl esterssuch as ethyl acetate, and methyl or ethyl propionate; amides such asN-methylpyrrolidinone, dimethylformamide and particularlydimethylacetamide; lower alkyl, for example C₂₋₄ nitriles such asacetonitrile; ethers such as glyme and diglyme and cyclic ethers such astetrahydrofuran and dioxane; tertiary amines, such asN-methylpyrrolidine and heterocyclic aromatic amines such as pyridine.and alcohols, most commonly the alcohol corresponding to the group R,for example methanol, ethanol, methoxyethanol, allyl alcohol orpropargyl alcohol.

The process of the present invention is often carried out at atemperature of from room temperature, such as about 25° C., up to thereflux temperature of the solvent employed. Temperatures above thenormal boiling point of the solvent employed can be employed if desiredby carrying out the process under super-atmospheric pressure conditions,for example in a sealed reaction vessel. Commonly, the temperature is inthe range of from 50 to 150° C.

The process commonly takes place over a period ranging from severalhours, for example from 4 to 12 hours, to several days, for example from1 to 2 days, depending on the reagents and reaction conditions employed.

When the compound of formula (1) comprises the base uracil, the uracilmoiety may be converted to a cytosine moiety. Similarly, the uracilmoiety comprised in the compound of formula (3) may also be converted toa cytosine moiety. The skilled man will recognise that a number ofdifferent techniques can be employed. Examples of such techniquesinclude:

-   a) the nitrophenyl route (see Miah et al, Nucleosides and    Nucletides, 1997, 16, pp 53-65) where for example the uracil    containing compound is reacted with chlorotrimethylsilane in    acetonitrile/1-methylpyrrolidine, then with trifluoroacetic    anhydride, followed by 4-nitrophenol. The 4-nitrophenol moiety is    then displaced with ammonia in aqueous dioxane to yield the    cytosine-containing compound; and-   b) the triazolation procedure, (see Divakar et al, J. Chem. Soc.    Perkin Trans. 1, 1982, 1171-6) where for example the uracil    containing compound is reacted with acetic anhydride in pyridine,    then, after work up, with phosphoryl chloride, 1,2,4-triazole and    triethylamine in acetonitrile to give the 4-triazolopyrimidine. The    triazole moiety is then displaced with ammonia in aqueous dioxane,    and acetyl groups removed to yield the cytosine-containing compound.

Protecting groups can be removed using methods known in the art for theparticular protecting group and function. For example, acyl protectinggroups, such as ethanoyl and benzoyl groups, can be removed by treatmentwith a solution of ammonia in an alcohol such as ethanol.

Benzoyl, pivaloyl and amidine groups can be removed by treatment withconcentrated aqueous ammonia.

Trityl groups present can be removed by treatment with acid, for examplea solution of dichloroacetic acid in dichloromethane. With regard to theoverall unblocking strategy an important consideration is that theremoval of trityl, often DMTr, protecting groups (‘detritylation’)should proceed without concomitant depurination when base B represents apurine, especially adenine. Such depurination can be suppressed byeffecting ‘detritylation’ with a dilute solution of hydrogen chloride atlow temperature, particularly ca. 0.45 M hydrogen chloride indioxane-dichloromethane (1:8 v/v) solution at −50° C. Under thesereaction conditions, ‘detritylation’ can be completed rapidly, and incertain cases after 5 minutes or less.

Silyl protecting groups may be removed by fluoride treatment, forexample with a solution of a tetraalkyl ammonium fluoride salt such astetrabutyl ammonium fluoride.

Fpmp protecting groups may be removed by acidic hydrolysis under mildconditions.

Compounds produced by the present invention may be incorporated in theassembly of a desired oligonucleotide by coupling with other nucleosidesor oligonucleotides (which may themselves have been prepared using thepresent invention) and such a process forms a further aspect of thepresent invention. The coupling processes employed are those known inthe art for the preparation of oligonucleotides.

The present invention is further illustrated, but not limited by, thefollowing Examples.

GENERAL EXPERIMENTAL DETAILS

Mps are uncorrected. ¹H and ¹³C NMR spectra were measured at 360.1 and90.6 MHz respectively, with a Bruker AM 360 spectrometer;tetramethylsilane was used as an internal standard. TLC was carried outwith Merck silica gel 60 F₂₅₄ pre-coated plates (Art 5715), which weredeveloped in solvent system A [CHCl₃-MeOH (85:15 v/v)]. Short columnchromatography was carried out on silica gel (Merck Art 7729).Acetonitrile and 1-methylpyrrolidine were dried by heating, underreflux, with calcium hydride and were then distilled.N,N-Dimethylacetamide (DMA) was dried by distillation over calciumhydride under reduced pressure. 2-Methoxyethanol was dried by heatingwith aluminium foil (1 g/250 ml), under reflux, and was then distilled.Diethyl ether was dried over sodium wire.

Preparation of 2,2′-Anhydro-1-β-D-arabinofuranosyluracil

Uridine (12.21 g, 50 mmol), diphenyl carbonate (11.79 g, 55 mmol),sodium hydrogen carbonate (0.219, 2.5 mmol) and dry DMA (10 ml) wereheated together, with stirring, at 100° C. After 5 h, the products werecooled to room temperature, and diethyl ether (100 ml) was added withstirring. After 2 hours, the colourless precipitate (11.70 g) wascollected by filtration and was washed with ether (2×50 ml). The solenucleoside constituent of the precipitated material was identified as2,2′-anhydro-1-β-D-arabinofuranosyluracil (calculated quantitativeyield, 11.31 g) by comparison with authentic material.

Preparation of 2′-O-(2-Methoxyethyl)uridine

Aluminium foil (3.64 g, 0.135 mol) and dry 2-methoxyethanol (135 ml)were heated, under reflux, for ca. 1 hr until all of the aluminium hadbeen consumed. Crude (see above)2,2′-anhydro-1-β-D-arabinofuranosyluracil (10.18 g, ca. 43.5 mmol) wasadded and the reactants were heated, under reflux, for 48 hours.Absolute ethanol (200 ml), followed by water (7.3 ml, 0.405 mol) andCelite were added to the cooled products. The resulting mixture washeated, under reflux, for 10 minutes and was then filtered. The residuewas washed with ethanol (3×100 ml). The combined filtrate and washingswere evaporated under reduced pressure to give a pale yellow solid. Thematerial was purified by short column choromatography on silica gel (70g): the appropriate fractions, which were eluted withdichloromethane-methanol (90:10 v/v), were evaporated under reducedpressure to give the title compound as a colourless solid (12.05 g, ca.91%).

Preparation of 2′-O-(2-Methoxyethyl)cytidine

2′-O-(2-Methoxyethyl)uridine (6.05 g, 20.0 mmol), 1-methylpyrrolidine(20 ml, 0.192 mol), chlorotrimethylsilane (7.6 ml, 59.9 mmol) and dryacetonitrile (100 ml) were stirred together at room temperature. After 1hour, the reactants were cooled to 0° C. (ice-water bath) andtrifluoroacetic anhydride (7.1 ml, 50.3 mmol) was added dropwise over 5minutes. After a further period of 30 minutes at 0° C., 4-nitrophenol(8.35 g, 60 mmol) was added to the stirred reactants which weremaintained at 0° C. After 3 hours, the products were poured intosaturated aqueous sodium hydrogencarbonate (200 ml), and the resultingmixture was extracted with dichloromethane (3×100 ml). The combinedorganic layers were dried (MgSO₄), and evaporated under reducedpressure. Concentrated aqueous ammonia (d 0.88, 20 ml) was added to astirred solution of the residue in dioxane (100 ml), contained in asealed flask that was then heated at 55° C. for 24 hours. The resultingyellow solution was concentrated under reduced pressure, and the residuewas evaporated with absolute ethanol (3×50 ml). The products werefractionated by short column chromatography on silica gel: theappropriate fractions, which were eluted withdichloromethane-methanol-triethylamine (93:7:0.5 to 90:10:0.5 v/v) wereevaporated under the reduced pressure to give the title compound as anoff-white solid (5.07 g 84%).

1. A process for the preparation of a compound of formula (1):

which comprises reacting a compound of formula (2):

with a compound of formula Al(OR)₃, under substantially anhydrousconditions wherein: X, and X¹ are each independently H or a protectinggroup; B is a nucleobase; and R is an alkyl, alkoxyalkyl, alkenyl, oralkynyl group, each of which may be substituted by one or more ofhalogen or amino substitutes; and L is a leaving group.
 2. A processaccording to claim 1, wherein the leaving group is selected from thegroup consisting of —OSO₂CH₃, —OSO₂CF₃, Cl, Br, I, O-Mesyl, O-BrosylO-Tosyl and the nucleobase, B, chemically bonded to the 2′-position, viaan oxygen or sulphur atom or a moiety of formula —NR^(X)—, wherein R^(X)is H or a C₁₋₆ or an aryl group.
 3. A process for the preparation of acompound of formula (3):

which comprises reacting a compound of formula (4)

with a compound of formula Al(OR)₃, under substantially anhydrousconditions wherein: X, and X¹ are each independently H or a protectinggroup; R¹ and R² are each independently H, alkyl, alkenyl, alkynyl, orhalogen; and R is an alkyl alkoxyalkyl, alkenyl, or alkynyl group, eachof which may be unsubstituted or substituted by one or more of halogenor amino substituents.
 4. A process according to claim 3, wherein R¹ andR² are both K or R¹ is C₁₋₄ alkyl, and R² is H.
 5. A process accordingto claim 1 or claim 3, wherein R is a C₁₋₄ alkenyl group, a C₁₋₄ alkylgroup, a C₁₋₄ alkyoxyC₁₋₄ alkyl group or a C₁₋₄ alkyl group.
 6. Aprocess according to claim 5, wherein R is a methoxyethyl group.
 7. Aprocess according to claim 1 for the preparation of a compound ofFormula (1) wherein B represents cytosine, or a substituted derivativethereof, which comprises: a) preparing said compound of Formula (1)wherein B represents uracil, or a substituted derivative thereof; and b)converting the uracil moiety to the equivalent cytosine moiety.
 8. Aprocess for the preparation of a compound of Formula (1)

wherein X and X¹ are each, independently, H or a protecting group; R isan alkyl, alkoxyalkyl, alkenyl, or alkynyl group, each of which may beunsubstituted or substituted by one or more of halogen or aminosubstituents; and B represents cytosine, or a substituted derivativethereof; which comprises a) preparing a compound of formula (3), by aprocess according to claim 3; and b) converting the uracil moiety to theequivalent cytosine moiety.
 9. A process for the preparation of aproduct oligonucleotide which comprises the coupling to a nucleoside oran oligonucleotide of a compound prepared by a process according to anyone of claim 1, 3, 7 or
 8. 10. A process according to claim 1 or claim3, wherein X and X¹ each represent H.
 11. A process according to claim 1or claim 3, wherein at least one of X and X¹ represent said protectinggroup.
 12. A process according to claim 11, wherein the protecting groupor groups are selected from the group consisting of acid labileprotecting groups, acid-labile acetal protecting groups; and baselabile-protecting groups.
 13. A process according to claim 1, whereinthe leaving group L is selected from the group consisting of —OSO₂CH₃,—OSO₂CF₃, Cl, Br, I, O-Mesyl, O-Brosyl, and O-Tosyl.
 14. A processaccording to claim 1, wherein the leaving group L is pyrimidine.